Centrifuge with removable core for scalable centrifugation

ABSTRACT

The present invention relates to a centrifuge apparatus. The centrifuge apparatus is operable at certain predetermined parameters depending upon a product to be separated and is useable with a plurality of rotor assemblies. For example, a first rotor assembly of said plurality of rotor assemblies includes a first core having a first core configuration which is contained within a rotor housing of the first rotor assembly to define a first volume capacity such that the product passing through the first rotor assembly having the first volume capacity during rotation of the first rotor assembly in the centrifuge apparatus achieves a first particle separation of the product. A second rotor assembly of said plurality of rotor assemblies includes a second core having a second core configuration which is contained with a rotor housing of the second rotor assembly to define a second volume capacity of such that product passing through the second rotor assembly having the second volume capacity during rotation of the second rotor assembly in the centrifuge apparatus achieves a second particle separation of the product. It is observed that the second particle separation is a linear change with respect to the first particle separation.

FIELD OF THE INVENTION

The present invention is directed to centrifuge equipment utilizing aremovable core which can be replaced with another core of differentdimensions to obtain directly linear scale process results for aparticulate protein separation and purification protocol. Moreparticularly, the invention provides a centrifuge rotor assemblycomprising means for adjusting the volume of the rotor assembly toaccommodate, for example, large-scale, pilot-scale and laboratory-scalecentrifugation needs.

Documents cited herein in the following text are incorporated byreference.

BACKGROUND OF THE INVENTION

In the biological and chemical sciences, there is often a need toseparate particulate matter suspended in a solution. In a biologicalexperiment, for example, the particles typically are cells, subcellularorganelles and macromolecules, such as DNA fragments. A centrifuge isroutinely used to perform the separation of these components from asolution.

The types of experiments that can be performed with a centrifuge arebased primarily on three major sedimentation (fractionation) protocols,namely, differential pelleting sedimentation (differentialcentrifugation), rate-zonal density-gradient sedimentation and isopycnicdensity-gradient sedimentation.

Basically, a centrifuge creates a centrifugal force field by spinning asolution containing suspended particles to be separated, thus causingthe suspended particles to separate from the solution. The sedimentationrate of a particle is a function of such factors as the molecular weightand density of the particle, the centrifugal field acting upon theparticle, and the viscosity and density of the solution in which theparticle is suspended.

A differential pelleting experiment is primarily used for thesedimentation of particles according to size. The material to befractionated is initially distributed uniformly throughout the samplesolution. A centrifuge tube filled with the sample solution is spun toproduce a centrifugal field which acts on the particles in the samplesolution. Eventually, a pellet is formed at the bottom of the tube whichis composed primarily of the larger particles present in the solution,but also includes a mixture of other smaller particles suspended in thesolution.

A rate-zonal separation protocol is used to improve the efficiency ofthe fractionation by separating the particles according to size.Rate-zonal sedimentation of particles relies on the property thatparticles of different sizes (and therefore different masses) willmigrate through a density-gradient at different rates when subjected toa centrifugal force.

The technique involves layering a sample containing the components ofinterest onto the top of a liquid column which is stabilized by adensity-gradient of an inert solute, such as sucrose. The maximumdensity of the gradient typically is less than the buoyant density ofthe components of interest, to allow migration of the components alongthe gradient. Upon centrifugation, the particles are driven down thegradient at a rate dependent upon factors including the mass and densityof each particle, the density of the gradient, and the centrifugalforces acting upon each particle. Generally, the more massive particleswill migrate at a faster rate than the lighter particles. With thepassage of time, numerous “zones” or “bands” of particles having similarmass will form. As the centrifugation continues, the widths of the zonesmeasured along the central axis of the centrifuge tube increase as wellas the separation between bands. In addition, the zones themselvesmigrate toward the bottom of the tube, and eventually will coalesce atthe bottom.

The third type of fractionation is another type of zonal separationcalled isopycnic density-gradient sedimentation, which relies ondifferences in the buoyant properties of the constituent particlesdispersed in a high density solution as the basis for separation of theconstituents. While centrifugation must proceed for a period of timesufficient to allow for banding, the protocol is an equilibriumtechnique in which separation essentially is independent of the time ofcentrifugation and of the size and shape of the constituents, althoughthese parameters do determine the rate at which equilibrium is reachedand the width of the zones formed at equilibrium.

There are two ways to prepare a solution for isopycnic separation. Asolute having a pre-formed high density-gradient is provided, in which asample containing the macromolecules is included. Subsequentcentrifugation of the preparation will cause the macromolecules of thesample to migrate through the high density solute, forming bands atpositions along the density-gradient corresponding to the buoyantdensity of each macromolecule. At each of these equilibrium positions,the buoyant force of the solute acting on a macromolecule is canceled bythe opposing forces of the centrifugal field. Alternatively, thesolution to be centrifuged may be prepared by mixing a solution of themacromolecules or particles of interest with a high density solute togive a uniform solution of both. In this case, the density-gradientforms during the centrifugation, with the particles forming bands alongthe resulting gradient as described.

Present centrifuge systems provide users with an interface for selectingthe speed and duration of a centrifuge run. Additional parameters may beset, including a temperature setting for the run and the particularrotor to be used. Typically, a user will set up a centrifuge run firstby deciding which of the three types of centrifuge protocols isappropriate. Next, the user must determine the centrifugation speed andthe run-time and then set the centrifuge accordingly. Computing therun-speed and the run-time depends upon a number of factors, such as theselected centrifuge protocol, the sedimentation rate of the particlesand knowledge of the parameters of the rotor to be used. In the case ofdensity-gradient separations, namely, the rate-zonal and isopycnicprotocols, the gradient of the solute must be included in thecomputations as well. However, present centrifuges are not configured tobe scalable. In other words, users cannot utilize the same centrifugesystem to accommodate the varying volumetric sizes required forlaboratory scale, pilot-scale and large scale needs.

Centrifugation separations are based on particle movement in an appliedcentrifugal field and the parameters of density, molecular weight andshape will affect this separation. For instance, classification ofcentrifugation techniques has split the field into preparative andanalytical methods for the range of sub-cellular particles, single cellorganisms, viruses, and macromolecules.

Analytical centrifugation has been used to obtain information regardingmolecular structure, interactions of molecules, and to give an initialestimation of molecular types in a new preparation. Preparativecentrifugation utilizes the same separation principles of analyticalcentrifugation to achieve a bulk manufacture of biological materials foruse in parenteral or diagnostic processes.

Zonal rotor assemblies have been used for many years and considerableliterature is available on the subject. Information about zonal rotorsis included in most purification handbooks and biochemistry texts.Specific information can be found in Anderson, An Introduction toParticle Separations in Zonal Centrifuges (National Cancer InstituteMonograph No. 21, 1966); Anderson, Separation of Sub-Cellular Componentsand Viruses by Combined Rate and Isopycnic Zonal Centrifugation(National Cancer Institute Monograph No. 21, 1966); and, Anderson,Preparative Zonal Centrifugation, in Methods of Biochemical Analysis(1967), all of which are incorporated herein by reference.

Typically, the zonal rotor assembly has an outer cylinder for containingthe product and the outer cylinder is subdivided with unitarily formedinterceptive cross-bars (sometimes referred to as fins or vanes) whichextend and are attached to the bowl and are not exposed therefrom.

The zonal rotor assembly is made, for example, of titanium and asaforementioned in a one piece construction of the outer cylinder andcross bars with a lid, which provides the strength needed to withstandthe high gravitational forces necessary for the ultracentrifugation upto 150,000×g. Two general formats of zonal rotors were developed,commonly known in the art as the bowl type and the tubular type rotorassemblies.

The bowl type rotor assembly, for example, the Ti-15 (Beckman CoulterInc.), is a wide squat bowl-shaped rotor assembly and can typically beused to 90,000×g in a batch mode operation. The same type of rotor wasmanufactured by Beckman Coulter to enable continuous flow operation.

Tubular assembly rotors were developed by Electro-Nucleonics (now AWI)and Hitachi Koki Co. (distributed by Kendro) and are long and tubular inshape and generate gravitational force up to 121,000×g. A centrifugeincorporating a tubular rotor assembly is described by Hsu, Separationand Purification Methods, 5(1), 51-95 (1976), which is incorporatedherein by reference.

Density gradient ultra-centrifugation using a zonal rotor assembly as apreparative methodology has been used widely to fractionate differentsubstances or materials, included but not limited to animal, plant andbacterial cells, viral particles, lysosomes, membranes andmacromolecules in a variety of processes. As an example, its applicationis of particular significance in the commercial preparation of virusesfor vaccine and immuno-therapy products in both batch and continuousflow zonal modes. These methods are traditionally used to purifyinfluenza virus for vaccines. In addition, many other uses for the zonalcentrifuge tubular or batch types have been documented, see Cline,Progress in Separation and Purification (1971), which is incorporatedherein by reference.

Although the small scale tubular rotor assemblies in the art provide anadequate separation, they are not suited for linear scale separationsbecause of, for example, differences in path length and wall affects(see Rickwood, Preparative Centrifugation: A Practical Approach, 1992,incorporated herein by reference).

Density gradient ultra-centrifugation, a type of zonal separation,enables sufficient and rapid purification of macromolecules for initialprotein characterization studies without the requirement of a lengthyprocess of development and optimization of a chromatography technique.Furthermore, density gradient ultra-centrifugation remains a preferredcost-effective route for the commercial separation of large particulateviruses and vaccines.

Most zonal separation is undertaken using density gradients which areloaded into the rotor assembly prior to loading the fluid containing theparticle product. Particle separation occurs in the gradient ofincreasing density. The particles eventually band isopycnically in thezones where the gradient density equals the particles' buoyant density.

A disadvantage of current zonal separation centrifuge systems is thatthey are not linearly scalable. In other words, a user cannot scale upor down, for separations of different volumes or quantities, e.g., fromlaboratory scale to pilot scale to industrial scale or from industrialscale pilot scales to laboratory scale, using the same centrifugationsystem.

A need exists in the art, therefore, to use the same centrifuge systemfor sedimentation processes of different volumes or quantities e.g.,large-scale, pilot-scale and laboratory-scale processes. In the knownart, if a centrifuge system was used in a laboratory scale process, itcould not be used in a pilot or large scale process. Each processrequired different centrifuge machinery. Each case also required thedetermination of new process parameters in order to achieve the sameseparation characteristics. In contrast to the prior art, the presentinvention provides a method and apparatus for adjusting the volume ofthe rotor assembly so the same centrifuge systems can be used forsedimentation processes of multiple scales while maintainingsubstantially the same separation characteristics for each process. In apreferred embodiment, the volume of the rotor assembly is adjusted byinterchanging different sized and configured core assemblies within theouter cylindrical rotor housing, thus affording a considerableimprovement to the current range of centrifugation products.

OBJECTS OF THE INVENTION

Therefore, it is an object of the invention to provide an improvedcentrifuge apparatus and process which avoids the aforementioneddeficiencies of the prior art.

It is an object of the invention to provide a centrifuge apparatus andprocess in which the volume of the product sample centrifuged can bescaled up or down while maintaining substantially the same selectedseparation parameters of the process.

It is an object of the invention to provide a centrifuge apparatus andprocess in which the volumetric capacity of the rotor assembly of thecentrifuge can be varied or changed to accommodate different volumes ofproduct sample to be centrifuged.

It is another objective of the invention to provide replaceable cores ofdifferent sizes which can be utilized in the same centrifuge apparatusto change the volumetric capacity of the rotor assembly to allow scaleups or scale downs of product sample to be centrifuged withoutsubstantially altering selected separation parameters such assedimentation path, residence path and flow dynamics.

Various other objects, advantages and features of the present inventionwill become readily apparent from the ensuing detailed description andthe novel features will be particularly pointed out in the appendedclaims.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention, a centrifugeapparatus is operable at certain predetermined parameters depending upona product to be separated and is useable with a plurality of rotorassemblies wherein a first rotor assembly of said plurality of rotorassemblies includes a first core having a first core configuration whichis contained within a rotor housing of the first rotor assembly todefine a first volume capacity such that the product passing through thefirst rotor assembly having the first volume capacity during rotation ofthe first rotor assembly in the centrifuge apparatus achieves a firstparticle separation of the product, and a second rotor assembly of saidplurality of rotor assemblies includes a second core having a secondcore configuration which is contained with a rotor housing of the secondrotor assembly to define a second volume capacity such that productpassing through the second rotor assembly having the second volumecapacity during rotation of the second rotor assembly in the centrifugeapparatus achieves a second particle separation of the product which isa linear change with respect to the first particle separation.

In accordance with a further embodiment of the present invention, acentrifuge system includes a rotor assembly which contains the productsample that is to be centrifuged. The rotor assembly includes an outerrotor housing and a core which freely rotates to create the centrifugalforce that separates the desired particles from the product sample. Therotor assembly capacity is essentially the capacity of the rotorassembly with the core installed in the rotor housing. In the invention,the rotor assembly capacity is variable to accommodate correspondinglydifferent volumes of product sample without substantially changingselected separation parameters, such as a rotational speed andgravitational force, as the rotor assembly capacity is varied.

In accordance with yet another embodiment, a centrifuge apparatus isoperable at certain predetermined parameters depending upon a product tobe separated and is usable with a plurality of rotor assemblies whereina first rotor assembly of said plurality of rotor assemblies has a firstresidence length such that the product passing through the first rotorassembly during rotation thereof in the centrifuge apparatus achieves afirst particle separation of the product and a second rotor assembly ofsaid plurality of rotor assemblies has a second residence length suchthat the product passing through the second rotor assembly duringrotation thereof in the centrifuge apparatus achieves a second particleseparation of the product which is a linear change with respect to thefirst particle separation.

In accordance with still another embodiment, the rotor assembly capacityis changed by providing more than one core for the rotor assembly. Eachcore has a different configuration from the other core(s). The use ofone core in the rotor assembly will result in a rotor assembly capacitywhich is different from the rotor assembly capacity when another core isutilized. In one aspect of the invention, the different sized orconfigured cores can be used to allow the user to operate the centrifugein different volumes of product samples. In a further aspect of theinvention, the cores can be configured so that use of the differentcores not only changes the capacity of the rotor assembly but alsosubstantially maintains selected separation parameters in the centrifugeprocess.

In accordance with a further embodiment, the rotor assembly includes anouter rotor housing which is formed as a hollow cylinder with threadedend caps to form the outer body of the rotor assembly. An inner core isadapted to be contained within the outer body so as to create a flowpath of particles within the rotor assembly. The inner core includestubular channels for fluid flow and a plurality of fins extend radiallyfrom the center core and prevent mixing of the particles during use. Aswill be explained in more detail below, the size and configuration ofthe inner core and the fins integrally formed thereto can be altered tochange the volume and hence the capacity of the rotor assembly.Moreover, the residence capacity of the rotor assembly can be changed soas to provide linear separation of the particles within the rotorassembly.

The present invention further provides a method for rapidly changing thevolume capacity during centrifugation but maintains performanceparameters, such as the rotational speed and gravitational force of therotor assembly, irrespective of the volume capacity of the rotorassembly. The method includes the steps of operating a centrifugeapparatus at certain predetermined parameters depending upon a productto be separated, rotating a first rotor assembly having a firstresidence length in the centrifuge apparatus, passing the productthrough the first rotor assembly during rotation thereof to achieve afirst particle separation of the product, substituting the first rotorassembly in the centrifuge apparatus with a second rotor assembly havinga second residence length and rotating the second rotor assembly withinthe centrifuge apparatus, passing the product through the second rotorassembly during rotation thereof to achieve a second particle separationof the product which is linear with respect to the first particleseparation.

In another aspect of the present invention, the method includes thesteps of operating a centrifuge apparatus at certain predeterminedparameters depending upon a product to be separated, placing a firstcore having a first core configuration in a rotor housing to define afirst rotor assembly having a first volume capacity, rotating the firstrotor assembly having first volume capacity in the centrifuge apparatusso as to achieve a first particle separation of the product,substituting a second core having a second core configuration within therotor housing to define a second rotor assembly having a second volumecapacity, rotating the second rotor assembly having the second volumecapacity in the centrifuge apparatus so as to achieve a second particleseparation of the product which is linear with respect to the firstparticle separation. In this aspect of the invention, the volumecapacity of the rotor assembly can be changed by varying the size, crosssection and number of rotor fins which extend radially outwardly fromand are integrally formed with the core.

Therefore, the present invention provides a centrifuge apparatus andprocess in which the volumetric capacity of the rotor assembly can bevaried or changed to accommodate different volumes of product sample tobe centrifuged. In addition, the present invention provides forreplaceable cores with different fin configurations which can be used inthe same centrifuge apparatus to change the volumetric capacity of therotor assembly to allow scale up or scale down of the product sample tobe centrifuged without substantially altering selected separationparameters.

These and other embodiments of the invention are provided in or areobvious from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings in which:

FIG. 1 is a front elevational view of a centrifuge apparatus including apreferred embodiment of a centrifuge rotor assembly in accordance withthe teachings of the present invention.

FIG. 2 a is a front cross-sectional view of a preferred embodiment of arotor assembly to be rotated in the centrifuge apparatus of FIG. 1.

FIG. 2 b is a front cross-sectional view of a preferred embodiment of arotor assembly to be rotated in the centrifuge apparatus of FIG. 1.

FIG. 3 a is a front perspective view of a core to be contained withinthe cylindrical rotor housing of FIG. 2 a.

FIG. 3 b is a side elevational view of a core to be contained within thecylindrical rotor housing of FIG. 2 a.

FIG. 4 is a front elevational view of the core of FIG. 3 a illustratingthe flow path of product in the core assembly.

FIG. 5 is a graphic representation of the process steps undertaken inzonal centrifugation utilizing the rotor assembly of FIG. 2 a.

FIG. 6 is a side elevational view of another preferred embodiment of arotor assembly to be rotated in the centrifuge apparatus of FIG. 1 to beused in large scale volume centrifugation applications.

FIG. 7 is a chart representing the variables involved in calculating thevolume available for centrifugation utilizing the rotor assembly of FIG.6.

FIG. 8 is a side elevational view of a preferred embodiment of a coreassembly to be contained within the rotor housing of the rotor assemblyof FIG. 2 a to be used in large scale volume centrifugationapplications.

FIG. 9 is a chart representing the variables involved in calculating thevolume available for centrifugation utilizing the rotor assembly of FIG.8.

FIG. 10 is a side elevational view of another preferred embodiment of acore assembly to be contained within the rotor housing of the rotorassembly of FIG. 2 a to be used in large scale volume centrifugationapplications.

FIG. 11 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 10.

FIG. 12 is a side elevational view of another preferred embodiment of acore assembly to be contained within the rotor housing of the rotorassembly of FIG. 2 a to be used in large scale volume centrifugationapplications.

FIG. 13 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 12.

FIG. 14 is a side elevational view of another preferred embodiment of acore assembly to be contained within the rotor housing of the rotorassembly of FIG. 2 a to be used in large scale volume in centrifugationapplications.

FIG. 15 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 14.

FIG. 16 is a side elevational view of yet another embodiment of a rotorassembly to be rotated in the centrifuge apparatus of FIG. 2 b to beused in pilot and laboratory scale volume centrifugation applications.

FIG. 17 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 16, wherein the volume is approximately 1600 ml.

FIG. 18 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 16, wherein the volume is approximately 800 ml.

FIG. 19 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 16, wherein the volume is approximately 400 ml.

FIG. 20 is a side elevational view of a preferred embodiment of a coreassembly to be contained within the rotor housing of FIG. 2 b to be usedin pilot and laboratory scale volume centrifugation applications.

FIG. 21 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 20.

FIG. 22 is a side elevational view of another preferred embodiment of acore assembly to be contained within the rotor housing of FIG. 2 b to beused in pilot and laboratory scale volume applications.

FIG. 23 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 22.

FIG. 24 is a side elevational view of another preferred embodiment of acore assembly to be contained within the rotor housing of FIG. 2 b to beused in pilot and laboratory scale volume applications.

FIG. 25 is a chart representing the variables involved in calculatingthe volume available for centrifugation utilizing the rotor assembly ofFIG. 24.

FIGS. 26 a-d are charts representing the analyses performed on the postbanding fractions to measure scalability and linearity of four differentcore assemblies.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The embodiments of the present invention can be used to performseparations and, more particularly, separations of liquid, fluid and/orparticulate matter. The separation techniques include but are notlimited to density gradients on a continuous or batch basis, pelleting,rate zonal separations and gradient resolubilization.

The present invention provides for a centrifuge rotor assemblycomprising means for adjusting the volume of the rotor assembly toaccommodate, for example, large-scale, pilot scale and laboratory scaleseparations. The separations utilizing the present invention are bothscalable and linear. Scalability is the ability to go from one volume ofproduct to another volume of product without significant changes to thecentrifuge protocol. Linearity is the ability for the centrifuge toseparate different density materials to yield the same purificationresults and/or concentration. The present invention provides, therefore,a centrifuge apparatus and process in which the volume of the productsample centrifuged can be scaled up or down while maintainingsubstantially the same selected separation parameters of the process; acentrifuge apparatus and process in which the volumetric capacity of therotor assembly of the centrifuge can be varied or changed to accommodatedifferent volumes of product sample to be centrifuged; and replaceablecores of different sizes which can be utilized in the same centrifugeapparatus to change the volumetric capacity of the rotor assembly toallow scale ups or scale downs of product sample to be centrifugedwithout substantially altering selected separation parameters such assedimentation path, residence path and flow dynamics. As will be seen inthe Examples that follow, formation of equivalent gradients among thelarge-scale and pilot scale rotor assemblies; equivalent productseparation at the iso-dense layer in each scale of rotor assembly; andequivalent product peak shape in the gradient for each scale rotorassembly indicate that scalability and linearity are achieved.

Specifically, the present invention is directed to a centrifugeapparatus that is operable at certain predetermined parameters dependingupon a product to be separated. The centrifuge apparatus is useable witha plurality of rotor assemblies. For example, a first rotor assembly ofsaid plurality of rotor assemblies may include a first core having afirst core configuration which is contained within a rotor housing ofthe first rotor assembly. The first core defines a first volumecapacity. Thus, when a product passes through the first rotor assemblyhaving the first volume capacity during rotation of the first rotorassembly in the centrifuge apparatus, a first particle separation of theproduct is achieved. A second rotor assembly of said plurality of rotorassemblies includes a second core having a second core configurationwhich is contained with a rotor housing of the second rotor assembly todefine a second volume capacity. Thus, a product passing through thesecond rotor assembly having the second volume capacity during rotationof the second rotor assembly in the centrifuge apparatus achieves asecond particle separation of the product. The second particleseparation is linear with respect to the first particle separation.

In a preferred embodiment, the present invention contemplates that therotor housing of the first and the second rotor assemblies to be thesame. In other words, the rotor housing has the same residence length.

Further, the centrifuge apparatus of the present invention is operableat certain predetermined parameters and is usable with a plurality ofrotor assemblies, wherein a first rotor assembly of said plurality ofrotor assemblies has a first residence length such that the productpassing through the first rotor assembly during rotation thereof in thecentrifuge apparatus achieves a first particle separation of theproduct. A second rotor assembly of said plurality of rotor assemblieshas a second residence length such that the product passing through thesecond rotor assembly during rotation thereof in the centrifugeapparatus achieves a second particle separation of the product. Thesecond particle separation is linear with respect to the first particleseparation.

The present invention also contemplates a method for achieving linearscale separation of particles of a product during centrifugation. Acentrifuge apparatus is operated at certain predetermined parametersdepending upon a product to be separated. A first core having a firstcore configuration is placed in a rotor housing to define a first rotorassembly having a first volume capacity. The first rotor assembly havingthe first volume capacity in the centrifuge apparatus is rotated,whereby the product is passed through the first rotor assembly duringrotation. This first rotation achieves a first particle separation ofthe product. A second core having a second core configuration issubstituted for the first core within the rotor housing to define asecond rotor assembly having a second volume capacity. This second rotorassembly is rotated, during which the product is passed through thesecond rotor assembly during rotation thereof, thereby achieving asecond particle separation of the product. This second particleseparation is a linear change with respect to the first particleseparation.

A method for achieving a linear scale separation is also provided by thepresent invention. A centrifuge apparatus at certain predeterminedparameters depending upon a product to be separated is operated. A firstrotor assembly having a first residence length in the centrifugeapparatus is rotated, whereby the product passing through the firstrotor assembly during rotation achieves a first particle separation ofthe product. After the first particle separation, a second rotorassembly is substituted for the first rotor assembly. The second rotorassembly has a second residence length and the second rotor assembly isrotated within the centrifuge apparatus. During rotation, the productpasses through the second rotor assembly to achieve a second particleseparation of the product, the second particle separation being linearwith respect to the first particle separation.

The centrifuge apparatus of the present invention also comprises meansfor setting a number of parameters for the centrifugation. Adjustmentmeans are also provided for setting parameters and having one of a rotorassembly selected from among a plurality of rotor assemblies so as toenable volume capacity to be adjusted. The adjustment means enables, forexample, substitution of a rotor core of varying configurations withineach of said plurality of rotor assemblies.

The present invention further contemplates a rotor assembly rotatable ina centrifuge assembly for separating particles of a product passingtherethrough. The rotor assembly is provided with a rotor housing of adefined volume and a rotor core freely rotatable within the rotorhousing. The rotor core includes a plurality of product flowdistribution channels and a plurality of fins extending radiallytherefrom of a predetermined configuration to define a volume of thepredetermined rotor core.

A rotor core for a rotor assembly rotatable in a centrifuge assembly forseparating particles of a product passing through the rotor assembly isalso provided by the present invention. It is envisioned that the rotorcore includes a plurality of product flow distribution channels and aplurality of fins extending radially therefrom of a predeterminedconfiguration to define a predetermined volume of the rotor core.

Each rotor core of the plurality of rotor assemblies, as contemplated bythe present invention, includes a plurality of fins arranged in apredetermined manner. These fins are equidistantly spaced apart fromeach other and extend radially outward from the rotor core. The numberof fins contemplated to be placed on each core number from between 0 to36, preferably from between 0 to 6. Each rotor core also includes aplurality of product flow distribution channels.

I. Description of Centrifuge Apparatus and Basic Components

Reference is now made to the figures wherein like parts are referred toby like numerals throughout. FIG. 1 depicts centrifuge 100 according tothe present invention. Centrifuge 100 of the present invention may beutilized in a process for separating components of a product sample inwhich the volume of the product sample can be scaled up or down whilemaintaining substantially the same selected separation parameters of theprocess.

With particular reference to FIG. 1, centrifuge 100 includes a tankassembly 1 within which is housed a drive turbine and a rotor assembly2. The drive turbine is used to spin rotor assembly 2 at high speeds. Aswill be described in further detail below, the rotor assembly 2typically includes an outer rotor housing, two end caps and a core. Alift assembly 3 is provided to raise both the drive turbine and therotor assembly 2 from tank assembly 1. A console assembly 4 is providedwhich connects to tank assembly 1 and controls the critical functions ofcentrifuge 100 such as, for example, time and speed.

II. Description of Rotor Assembly

With reference to FIG. 2 a, useful for large scale separations andadapted to house cores with a residence length L₁ of, for example,approximately 30 inches, rotor assembly 2 is explained in furtherdetail. Rotor assembly 2 includes an outer rotor housing 5 and a core 6which is adapted to be disposed within outer rotor housing 5. Outerrotor housing 5 may be made of any material suitable in thecentrifugation art, preferably titanium. Core 6 may be made of anymaterial or blend of materials suitable in the centrifugation art, suchas, for example, a thermoplastic resin, titanium andpolyetheretherketone (PEEK). In a preferred embodiment, core 6 may beformed from a polymeric material such as, for example, a polyphenyleneether, or a blend of more than one polymeric material. A preferredpolyphenylene ether is available commercially from the General ElectricCompany and is sold under the trademark NORYL. Core 6 is substantiallycylindrical, but may be configured into any shape that can withstand thestress of centrifugation.

The rotor assembly 2 also includes top end cap 7 and bottom end cap 8.Teflon inserts 9 are adapted to be disposed between outer rotor housing5 and end caps 7 and 8 to seal the rotor assembly 2. Rotor assembly 2also includes O-rings 10, 11 and 12 to seal the rotor assembly 2.

With reference to FIG. 2 b, useful for laboratory and/or pilot scaleseparations and adapted to house cores with a residence length L₂ of,for example, approximately 15 inches, rotor assembly 2 a is explained infurther detail. The outer rotor housing 5 a and the core 6 a of therotor assembly 2 a can be formed of the same materials as the outerrotor housing 5 and core 6 of the rotor assembly 2 of FIG. 2.

III. Generalized Description of Core Assembly for Use in the RotorAssemblies of FIGS. 2 a and 2 b

Reference is now made to FIG. 3 a which is a front perspective view ofcore 6 in accordance with the teachings of the present invention whereinthe core 6 includes a plurality of fins 13 extending radially outwardfrom the length of the inner cylinder 110 of the core 6. It iscontemplated that core 6 typically comprises six fins 13, with thesefins being arranged equidistantly from each other. It is understood,however, that more or less than six fins may be used, for example from 0to 36 fins may be employed.

Additionally, reference is made to FIG. 3 b, wherein a side elevationalview of core 6 is depicted. As seen in FIG. 3 b, R1 represents thedistance from the center of core 6 to the inner cylinder 110. R2represents the distance from the center of core 6 to the outermost pointof fin 13. D1 represents the chord of the circle with a radius R1. D2represents the top width of fin 13. As seen in FIG. 3 b, the dimensionsof core 6 which are adjustable include, for example, D2 and radius R1.

From dimension D2, D1 is calculated so that the surface of fin 13 facingthe fluid to be centrifuged maintains an angle of, typically, 2 degreesfrom vertical. The length of fin 13 is defined by the angle and the tworadii (such as, for example, R1=2.143″ and R2=2.598″).

To determine the volume available for centrifugation when core 6 isdisposed within rotor assembly 2, the volume of core 6 typically needsto be calculated. With reference to FIG. 3B, the volume of core 6 can beapproximated as follows:V _(CORE) =V ₂ −V ₁−6V _(FIN)where:

-   -   V₂ is the volume of the outer cylinder of the core (with radius        R2),    -   V₁ is the volume of the inner cylinder of the core (with radius        R1),    -   V_(FIN) is the volume of a single fin of dimensions θ_(T), θ_(B)        and D2, and    -   V_(CORE) is the volume available for fluid during        centrifugation.

The volume of the outer cylinder of core 6 with a radius R2 (V₂) and thevolume of the inner cylinder of core 6 with a radius R1 (V₁) are easilydeterminable. The value of 6V_(FIN), however, is generally calculated asthe approximate volume occupied by fin 13. To this end, one wouldconsider a section defined by one-half fin 13. Thus, fin 13 isapproximated as a top-radiused trapezoidal section as shown below:

As D2 is a chord of the circle with a radius R2, the Top Fin Angle2θ_(T), wherein θ_(T) is the angle formed by one-half the top surface offin 13 in radians, can be calculated according to the law of cosines as:2θ_(T) =R 2 ² +R 2 ²−2(R 2)(R 2)cos(2θ_(T))or solving for θ_(T):θ_(T)=cos 1[(1−D 2 ²)/2R 2 ²)]/2

As the width across the bottom of fin 13 is typically such that an angleof approximately 2 degrees is maintained, and as the height of fin 13 istypically fixed, the end of the Fin Bottom (D1) is typically a fixeddistance beyond the end of the Fin Top to achieve the same angle. Inother words, D1=D2+the fixed distance (0.031″).

Further, as D1 is a chord of the circle with a radius R1, an angle2θ_(T) is calculated as:2(θ_(T)+θ_(B)Schwenk)=R 1 ² +R 1 ²−2(R 1)(R 1)cos(2(θ_(T)+θ_(B))),wherein θ_(B) is the angle formed by one-half the bottom fin surface inradians.

Thus, when the volume of core 6 is determined, the volume of the rotorassembly 2 may be increased and/or decreased depending on thecentrifugation protocol required by the user. Such an increase and/ordecrease in volume allows the centrifuge to be scaled either up or downfor industrial, pilot and laboratory uses, while maintainingsubstantially the same separation protocols.

With reference to FIG. 4, a cross-section of core 6 is illustratedwherein flow channel 14 is illustrated. Flow channel 14 provides a pathfrom the center 15 of core 6, in other words, from the point of productentry, to the chambers formed by fins 13. As seen in FIG. 4, the flowpath of a product to be separated enters rotor assembly through thecenter 15 of core 6. The product to be separated then flows through longthin tubular shafts 16 through core 6 and exits the centrifuge forcollection.

As shown in FIG. 5, the present invention is useful, for example, forzonal centrifugation. At step A, the density gradient 17 is loaded intothe rotor assembly 2 at rest. As the rotor assembly 2 is graduallyaccelerated, the gradient 17 reorients itself vertically along the wallsof rotor assembly 2 as shown in step B. Sample fluid 18 is pumped atstep C into rotor assembly 2 at one end 19 on a continuous flow basis.In step C, the sample particles 19 sediment radially into the gradient17 of increasing density. The sample particles 19 eventually band(isopycnically) in step D in those cylindrical zones where the gradientdensity equals a particle's buoyant density, commonly referred toiso-dense layers or zones. At the end of the run at step E, rotorassembly 2 is decelerated and the gradient 17 reorients to its originalposition at step F without disturbing the particle bands 20. The bandedparticles are now ready to be unloaded with rotor assembly 2 at rest.Fractions 21 are collected using air or water pressure and a smallperistaltic pump 22 to control flow at step G. Reorientation is welldescribed in many articles with respect to batch and continuous flowzonal rotors (see, e.g. Anderson, supra, 1967, which is incorporatedherein by reference).

In order to provide for a scale separation of reduced volume using thesame rotor assembly length, a change in configuration of core 6 tomaintain the flow path is necessary. The scale down in volume isachieved by maximizing the size of fin 13 of core 6 to reduce volumeradially, while at the same time substantially retaining the essentialsedimentation path and residence path of rotor assembly 2.

A further embodiment of the invention contemplates use of computers andsoftware for controlling the centrifuge and calculating thecentrifugation protocol. The software-driven control console assembly 4as seen in FIG. 1 gives the operator all operating parameters displayedin “real-time” on the control screen. Automated programs can also be runfrom pre-stored files, or manually through the control screen.

During each centrifuge run, on-line data monitoring and recording of setparameters, run parameters, and alarm status are made and aredown-loaded to the system memory. Such downloading may also be directedto an external data storage location.

A separation protocol typically involves knowledge of the physicalcharacteristics of the target protein; formation of the gradient; andthe calculation of run parameters. The physical characteristics of thetarget protein useful for defining a separation protocol include, forexample, the sedimentation coefficient (S_(20ω)) and buoyant density ofthe target protein. Such values are useful for reducing the number oftrial and error experiments. Otherwise, these can be estimated frompreliminary separations performed subsequently.

A separation protocol also typically involves formation of a gradient.The choice of gradient material depends on, for example, the product,impurity stabilities and product densities. Commonly used gradientmaterials include alkali metals, e.g. cesium chloride, potassiumtartrate, and potassium bromide. Although such materials may becorrosive, they create high densities with low viscosity.

CsCl is frequently used as a gradient material and can achieve highdensity (typically up to approx. 1.9 g/cm³). CsCl, however, can denaturecertain proteins. CsCl is also costly, may corrode aluminum rotorhousings, the steel of the seal assemblies and the rotor assemblyshafts. In addition it has been noted that free Cs⁺ ions are attractedto virus particles. Thus, binding of the virus particle to the toxicmetal ion may occur.

Another gradient material is potassium bromide. Although it can reachhigh densities, it can do so only at elevated temperatures, e.g. 25° C.Such elevated temperatures may be incompatible with the stability of theproteins of interest.

A preferred gradient material is sucrose. It is a cheaper gradientmaterial and utilizes a sufficient density range for most operations (upto approx. 1.3 g/cm³). The viscosity of a sucrose gradient allows forthe formation of a step gradient used for banding product, or,alternatively, to create a wide product capacity in the same rotor. Thestep gradient is the most efficient for continuous flow operation ifentry of the non-target protein is to be minimized.

The viscosity of sucrose is a desirable attribute to forming stepgradients for long periods of time in a continuous flow rotor. Bycontrast, a non-viscous solution, e.g. CsCl, may need the addition of ahigher-viscosity material, such as glycerol, to increase viscosity andminimize gradient erosion during the run.

The gradient may be loaded either as discontinuous steps or linearly.Loading the gradient as discontinuous steps or as linear gradientsallows for the use of a pre-formed gradient, which avoids extended runtimes to form the gradient. The reduced run time of the separation maybe useful for sensitive samples or small particulate proteins, whichtypically require longer run times to sediment sufficiently.

Loading discontinuous gradients may result in a discontinuous stepgradient, which provides for a better separation than a linear gradient.For batch zonal operations performed on a routine basis, the loading ofdiscontinuous step gradients is a simple and highly reproducibletechnique. A comparison of wide and narrow density gradient formats forcontinuous flow ultracentrifugation shows that a multi-step gradientforms a shallow gradient with high capacity for product accumulation,whereas a one-step gradient forms a steep gradient minimizingimpurities, while maintaining a relatively low capacity.

The shape of the gradient typically depends upon, for example, theinternal dynamics of rotor assembly 2. If a reorienting rotor assemblyis used, it is readily known that the acceleration and decelerationprofiles of the centrifuge should allow for reorientation withoutdisturbing the gradient. Further, the shape of the internal chambers inwhich the gradient reorients may cause a dispersion of the gradient. Ifa continuous flow rotor assembly is used, the generated flow can lead toan erosion of the gradient if there is instability in the system; and,upon longer or shorter run times, gradient shape will vary. It has beendiscovered that using the same centrifuge system is advantageous toscalability.

A separation protocol also typically involves the calculation of runparameters, such as the relative centrifugal force. The relativecentrifugal force (RCF) at the chosen speed is calculated by equation(1):RCF(g)=(1.421×10⁻⁵)(RPM)² d  (1)

-   -   d represents the core diameter (cm)    -   RPM represents revolutions per minute

This equation determines the force that a particular radius core canproduce. All cores of the same radius will typically produce the same gforce at the maximum diameter. This is typically relevant to pelleting.In gradient separations, however, there is banding of proteins ofinterest across the whole core radius which generates a range of gforces. The range of g force created is a function of the cross sectionpath length and, if the inner radius of two rotor assemblies differs,then the separation will differ also between the rotor assemblies. Thechoice of rotor assembly, therefore, depends on the composition of theproduct to be separated.

The efficiency of a rotor assembly is expressed as its K factor. The Kfactor provides an estimate of the time required to band a product at aset rotor assembly speed from an inner radius to a maximum radius. The Kfactor is usually supplied by the manufacturer of a centrifuge, but canalso be determined from equation (2): $\begin{matrix}{k = {\frac{{In}\quad( {r_{\max}/r_{\min}} )}{(\omega)^{2}} \times \frac{10^{13}}{3600}}} & (2)\end{matrix}$

-   -   (ω)=0.10472× revolutions per minute (RPM)    -   r_(max)=maximum radial distance from the center of rotation (cm)    -   r_(min)=minimum radial distance from the center of rotation (cm)

K is a specific value for a rotor assembly at a specific speed. K varieswith speed and could be calculated over the full operational speed ofthe rotor assembly. A low K factor indicates a rotor assembly's greaterefficiency.

If the sedimentation path remains constant rotor-to-rotor, then theseparation will remain scalable at different volumes. It is known,however, that rotor assemblies in the art differ greatly in the r_(min)r_(max) ranges.

The effect the K factor has on, for example, protein resolution dependson the proteins and the Svedberg Constant. For each protein product, theSvedberg constant can be determined using equation (3) but is oftensupplied by references to literature in a particular area of study. TheSvedberg value is a measure of the rate of movement in a rotor assemblyand is usually determined to estimate separations using analyticalrotors: $\begin{matrix}{S = {( {{1/W^{2}}R \times {{DR}_{a}/{DT}}} ) = \frac{L_{N}( {R_{\max} - R_{\min}} )}{W^{2}( {T_{2} - T_{1}} )}}} & (3)\end{matrix}$wherein:

-   -   G=Force    -   D=Diameter In Inches    -   L_(N)=Natural Log    -   R=Radius    -   R_(a)=Distance From The Axis    -   T=Time In Hours    -   T₂=End Time    -   T₁=Start Time    -   W=Molecular Weight

Once the Svedberg value is determined, the theoretical time for aparticular rotor assembly is calculated. The theoretical run time T iscalculated using equation (4).T=K/S _(20(ω))  (4)wherein:

-   -   T=time (hr)    -   k=rotor efficiency    -   S_(20(ω))=sedimentation coefficient

The theoretical runtime T, also known as the “residence time”, typicallyprovides for the theoretical minimum run time for a rotor assembly at aspecific K factor to ensure completion of product banding. There areother factors which can affect product bonding. Such factors includeaggregation, particle retention, denaturation, and the interaction withthe gradient. Particularly with the use of sucrose, an estimation mustbe made of the effect of viscosity in the gradient, which variescontinuously with increasing density. This is well known and has beentabulated (see McEwen, Analytical Biochemistry, 20:114-149, 1967,incorporated herein by reference).

The sedimentation coefficient (S_(20(ω))) of numerous particulateproteins and macromolecules are known and have been described in theliterature. Particulate proteins will tend to fall in the range of smallviruses 40S to 1500S.

If the K factor and the run time of a tubular rotor assembly are known,the run time of the zonal rotor assembly can be determined usingequation (5) without the need to calculate S_(20(ω)): $\begin{matrix}{t_{1} = {k_{1} \times {t_{2} \cdot k_{2}}}} & (5)\end{matrix}$wherein:

-   -   k₂ Efficiency of Rotor Assembly A    -   t₂ Run time of Rotor Assembly A    -   k₁=Efficiency of Rotor Assembly B    -   t₁=Run time of Rotor Assembly B

Typically, the protocol used at small scale and the preparative protocolto be derived thereon would use different speeds to run the separation.In order to determine the K factor at a different speed and, therefore,the time to sediment, equation (6) is used:K _(new) =k(Q _(max) /Q _(new))²  (6)wherein:

-   -   Q_(max) is the rotor maximum speed (rpm).    -   Q_(new) is the new rotor speed (rpm).

The present invention may also be used, for example, to pellet thetarget protein to the wall of rotor assembly 2; to sediment into a denseliquid; or to band in a gradient. Pelleting for example is suitable forextremely robust particles or cells. Sedimenting, for example, allowsfor recovery of the target protein with minimal loses due todenaturation. Banding in a gradient, for example, allows for removal ofimpurities.

The present invention may also be used for, for example, isopycnicbanding and rate zonal processes. Such processes may be used separatelyor may be combined to separate, for example, large heavy particles fromthe usually smaller impurities.

IV. Preferred Embodiments of the Core Assembly for Large ScaleProduction (FIGS. 6 to 15)

FIG. 6 through 15 are representative core assemblies in accordance withthe present invention which are designed for use in large-scaleproduction. Each of the cores 6 b-f of the respective core assemblies ofFIGS. 6, 8, 10, 12 and 14 are preferably made of NORYL™, but a skilledartisan would readily appreciate that any material suitable forcentrifugation may be used to manufacture the core.

In the embodiment shown in FIG. 6, core 6 b includes six fins 13 bequidistantly spaced apart and radially extending from inner cylinder110 b. The radii R1 and R2 of core 6 b are approximately equal to 2.145inches and 2.598 inches, respectively. The length of core 6 b isapproximately 30 inches. Utilizing formula V_(CORE)=V₂−V₁−6V_(FIN), andthe core dimensions represented by the chart of FIG. 7, the volumeavailable for centrifugation is approximately 3.2 liters.

With reference to another preferred core configuration in FIG. 8, core 6c includes six fins 13 c equidistantly spaced apart and radially extendfrom the inner cylinder 110 c. The radii R1 and R2 of the core 6 c areapproximately 0.825 inches and 2.598 inches, respectively. The length ofcore 6 c is approximately 30 inches. Utilizing formulaV_(CORE)=V₂−V₁−6V_(FIN), and the core dimensions set forth in the chartof FIG. 9, the volume available for centrifugation equals approximately8.4 liters.

With reference to another preferred core configuration of FIG. 10, core6 d includes six fins 13 d equidistantly spaced apart and radiallyextending from the inner cylinder 110 d. The radii R1 and R2 of the core6 d are approximately 2.145 inches and 2.598 inches, respectively. Thelength of core 6 d is approximately 30 inches. Utilizing formulaV_(CORE)=V₂−V₁−6V_(FIN), and the core dimensions set forth in FIG. 11,the volume available for centrifugation equals approximately 3.2 liters.

With reference to another preferred core configuration of FIG. 12, core6 e includes six fins 13 e equidistantly spaced apart and radiallyextending from the inner cylinder 110 e. The radii R1 and R2 of the core6 e are approximately 1.052 inches and 2.598 inches, respectively. Thelength of core 6 e is approximately 30 inches. Utilizing formulaV_(CORE)=V₂−V₁−6V_(FIN), and the core dimensions set forth in FIG. 13,the volume available for centrifugation equals approximately 8.0 liters.

With reference to another preferred core configuration of FIG. 14, core6 f includes radii R1 and R2 approximately 2.561 inches and 2.598inches, respectively. The length of core 6 f is approximately 30 inches.Utilizing formula V_(CORE)=V₂−V₁−6V_(FIN), and the core dimensions setforth in FIG. 15, the volume available for centrifugation equalsapproximately 0.3 liters.

The above figures demonstrate that, given a core with a fixed length,such as, for example, 30 inches, the volume available for centrifugationmay be altered by manipulating the dimensions and, thereby, the volumeof fins 13 of the core assembly. As will be demonstrated below,formation of equivalent gradients among the large-scale and pilot scalerotor assemblies; equivalent product separation at the iso-dense layerin each scale of rotor assembly; and equivalent product peak shape inthe gradient for each scale rotor assembly indicate that scalability andlinearity are achieved.

V. Preferred Embodiments of the Core Assembly for Small-Scale Production(FIGS. 16 to 25)

FIGS. 16 to 25 are representative core assemblies in accordance with thepresent invention which are designed for use in small-scale, e.g., pilotand laboratory scale, production. Each of the cores 6 g-j of therespective core assemblies of FIGS. 16, 18, 20, 22 and 24 are preferablymade of NORYL™, but a skilled artisan would readily appreciate that anymaterial suitable for centrifugation may be used to manufacture thecore.

In the embodiment shown in FIG. 16, core 6 g includes six fins 13 gequidistantly spaced apart and radially extending from inner cylinder110 g. The radii R1 and R2 of core 6 g are approximately 2.145 inchesand 2.598 inches, respectively. Core 6 g is preferably made of NORYL™,but a skilled artisan would understand that any material suitable forcentrifugation may be used to manufacture the core. The length of core 6g is approximately 15 inches. Utilizing formula V_(CORE)=V₂−V₁−6V_(FIN),and the dimensions of core 6 g represented by the chart of FIG. 17,wherein, for example, theta-T equals 0.0160 radians and theta-B equals0.0106 radians, the volume available for centrifugation equalsapproximately 1.6 liters. Further, utilizing formulaV_(CORE)=V₂−V₁−6V_(FIN), and the dimensions of core 6 g represented bythe chart of FIG. 18, wherein, for example, theta-T equals 0.2521radians and theta-B equals 0.0625 radians, the volume available forcentrifugation of core 6 g of FIG. 16 equals approximately 0.8 liters.Also, utilizing formula V_(CORE)=V₂−V₁−6V_(FIN), and the dimensions ofcore 6 g represented by the chart of FIG. 19, wherein, for example,theta-T equals 0.3640 radians and theta-B equals 0.0899 radians, thevolume available for centrifugation of core 6 g of FIG. 16 equalsapproximately 0.4 liters.

With reference to another preferred core configuration of FIG. 20, core6 h includes six fins 13 h equidistantly spaced apart and radiallyextending from the inner cylinder 110 h. The radii R1 and R2 of the core6 h are approximately 2.145 inches and 2.598 inches, respectively. Thelength of core 6 h is approximately 15 inches. Utilizing formulaV_(CORE)=V₂−V₁−6V_(FIN), and the core dimensions set forth in the chartof FIG. 21, the volume available for centrifugation equals approximately1.6 liters.

With reference to another preferred core configuration of FIG. 22, core6 i includes six fins 13 i equidistantly spaced apart and radiallyextending from the inner cylinder 110 i. The radii R1 and R2 of the core6 i are approximately 1.052 inches and 2.598 inches, respectively. Thelength of core 6 i is approximately 15 inches. Utilizing formulaV_(CORE)=V₂−V₁−6V_(FIN), and the core dimensions set forth in the chartof FIG. 23, the volume available for centrifugation equals approximately3.9 liters.

With reference to another preferred core configuration of FIG. 24, core6 j includes radii R1 and R2. The radii R1 and R2 are approximately2.561 inches and 2.598 inches, respectively. The length of core 6 j isapproximately 15 inches. Utilizing formula V_(CORE)=V₂−V₁−6V_(FIN), andthe core dimensions set forth in the chart of FIG. 25, the volumeavailable for centrifugation equals approximately 0.1 liters.

The above figures demonstrate that, given a core with a fixed length,such as, for example, 15 inches, the volume available for centrifugationmay be altered by manipulating the dimensions and, thereby, the volumeof fins 13.

DETAILED EXAMPLES

The following examples are set forth to illustrate examples ofembodiments in accordance with the invention, it is by no way limitingnor do these examples impose a limitation on the present invention.

The following examples demonstrate that scalability and linearity areachieved using the embodiments of the invention while maintaining thesedimentation path, residence path, and flow dynamics. In particular,the following examples demonstrate, for example, that a centrifugeapparatus operable at certain predetermined parameters depending upon aproduct to be separated and useable with a plurality of rotor assemblieswherein a first rotor assembly of said plurality of rotor assembliesincludes a first core having a first core configuration which iscontained within a rotor housing of the first rotor assembly to define afirst volume capacity such that the product passing through the firstrotor assembly having the first volume capacity during rotation of thefirst rotor assembly in the centrifuge apparatus achieves a firstparticle separation of the product, and a second rotor assembly of saidplurality of rotor assemblies includes a second core having a secondcore configuration which is contained with a rotor housing of the secondrotor assembly to define a second volume capacity such that productpassing through the second rotor assembly having the second volumecapacity during rotation of the second rotor assembly in the centrifugeapparatus achieves a second particle separation of the product which isa linear change with respect to the first particle separation.

Further, the following examples demonstrate that scalability andlinearity are achieved because, for example, formation of equivalentgradients among the large-scale and pilot scale rotor assemblies wasobserved; equivalent product separation at the iso-dense layer in eachscale of rotor assembly was observed; and equivalent product peak shapein the gradient for each scale rotor assembly was observed. In otherwords, scalability and linearity are achieved by, for example, operatinga centrifuge apparatus at certain predetermined parameters dependingupon a product to be separated; placing a first core having a first coreconfiguration in a rotor housing to define a first rotor assembly havinga first volume capacity; rotating the first rotor assembly having thefirst volume assembly having the first volume capacity in the centrifugeapparatus and passing the product through the first rotor assemblyduring rotation thereof so as to achieve a first particle separation ofthe product; substituting a second core having a second coreconfiguration within the rotor housing to define a second rotor assemblyhaving a second volume capacity; and rotating the second rotor assemblyhaving the second volume capacity in the centrifuge apparatus andpassing the product through the second rotor assembly during rotationthereof so as to achieve a second particle separation of the productwhich is a linear change with respect to the first particle separation.

Example 1 Preparation of Sucrose

Sucrose crystals (Life Technologies Inc.) were weighed using a top panbalance (two decimal places accuracy) in aliquots of 100 g. Lab waterwas heated to 60° C. using a heated stir plate. Temperature was measuredusing a 0-100° C. thermometer. At 60° C. the sucrose was gradually addedto the water.

1 or 2 liter lots of sucrose were made and pooled, and stock solutionsof 60% w/w sucrose were made. The sucrose density was checked with arefractometer for each lot to maintain consistency to within 60±2%sucrose.

Example 2 Preparation of Beads

Microsphere beads (Bangs Labs Inc.) were diluted in water atconcentrations for spectrophotometric analysis. The analysis would beperformed on the gradient fractions collected after separation.

Dilutions were made to give an absorbance peak of 1 AU (absorbance unit)at 280 nm. A scan peak of measurement at approximately 265 nm was chosenfor analysis of the beads. This proved to be too concentrated to load tothe system and a peak of 0.04 OD 280 nm was used. The UV analyses wererun at 265 nm, 280 nm and 320 nm. The 280 nm analysis typically showedless variation due to light sensitivity than the analysis at 265 nm. The320 nm analysis was used to show any light scattering caused bycontaminants. A ratio can be calculated between the three analyses tocheck for contamination of the product to be analyzed. Dilutions weremade using p1000 and p200 Gilson pipettes.

A Perkin Elmer Xpress UV spectrophotometer system was used with 1 cmpath, 2 ml volume cuvettes. A double beam was used with a blank lane anda test lane. The system was run for base line against water beforestarting. A calibration was made using the following calibration values:60% w/w sucrose, RI 1.4418 @ 20 C, 1.2865 g/cm³ @ 20 C, MWT 342.3, 771.9mg/ml and 2.255 M. All samples were diluted to 0 to 1 absorbance unitfor reading. Dilutions were made with water.

Sucrose concentration was measured using the Atago N-2E (Cole PalmerInstrument Co.) hand held refractometer. To check for linearity beforeuse, a dilution series was made in sucrose.

Example 3 Rotor Assembly and System Setup

The assembly of both the large scale and pilot-scale ultracentrifugesfollowed similar protocols. Some of the operational procedures differeddue to the different control consoles. Seal assemblies and rotorassemblies were cleaned with water. Ethanol spray was used to removevisible particulate matter from all surfaces. The rotor assemblies wereloaded to the centrifuge system, connections made, subsystems checked,and system started according to the instruction manuals.

In both the large scale and pilot scale systems, the rotor assembly tobe tested was filled with water using a peristaltic pump. In addition, acontainer with a further 2× rotor volume of water was attached to thepump inlet and recirculated from the centrifuge top outlet. This allowedfor water circulation during the start up phase. In both centrifugesystems, the instruction manuals were followed to perform the followingsteps: the pump was set to deliver approximately 300 ml/min to therotor; system was run in manual mode to 10,000 rpm; system was run withbuffer from top to bottom and bottom to top at 10,000 rpm to remove anybubbles; and system was run down to 0 rpm with buffer flow continuing inthe bottom to top direction.

Example 4 Gradient Loading and System Run

Sucrose solution was loaded from the bottom inlet of the system via aperistaltic pump. The sucrose solution was flushed through the pump to aTee-piece within 50 cm of the bottom inlet of the rotor. At this pointthe rotor outlet was diverted to a measuring cylinder appropriate to thevolume to be displaced.

The sucrose solution was then introduced into the rotor assembly to fillhalf the volume of the rotor assembly. The volume loaded was measured asthe volume of water displaced from the top of the rotor. When loaded,the rotor bottom inlet was closed, the sucrose flushed from the inletpump to the Tee-piece line.

In both the large-scale and pilot scale systems, the run was started inan auto ramp mode. This provided a smooth regulated acceleration toallow reorientation of the sucrose gradient without disturbance of thelayers of sucrose added while stationary to the rotor.

The speed was set to 3,500 rpm. When this speed was reached, the pumpwas set to run from top to bottom at the product flow rate (calculatedfor each run). Once any residual sucrose was displaced, the speed wasset to 40,500 rpm. At the maximum speed the product inlet was divertedto the test sample. When the entire test sample was loaded the productpump was diverted to the circulating water.

The test sample was left to band for a minimum 30 minutes with a minimalflow rate. Product flow was stopped and the deceleration with brakeapplied in the Auto ramp mode. At 0 rpm the product was collected.

Example 5 Product Collection

A product pump was set to remove the volume of liquid from the rotorbottom inlet and dispense to containers. Air was allowed to enter thetop inlet of the rotor. The rotor volume was divided into 30 fractions.Fraction collection was made by eye for determination of volume bycomparison to two standard solutions placed on either side of thefraction to be collected. Collected product was immediately analyzed fordensity and absorbance. Fractions were stored at room temperature beforedisposal.

Example 6 Product Analysis

On collection, product fractions were measured for absorbance at A₃₂₀,A₂₈₀ and A₂₆₅. For samples with greater than 1 AU in the sample, adilution was made and a second reading taken. The refractive index wasmeasured at room temperature with no dilution to sample. No adjustmentwas made for temperature in the display of results.

Example 7 Analysis of Data

Data collected was plotted as graphs of density versus absorbance. Theslope of the sucrose was determined, as well as the peak A₂₆₀ sucrosedensity.

Example 8 Rotor Selection

The rotor assemblies tested comprised cores having volumes of 3,200 ml,1,600 ml, 800 ml and 400 ml. The cores were machined from NORYL™, testedas PS280014 (AWI ISO procedure), and then made into high flow format.Details of cores chosen for experimentation Volume R_(min) R_(max) Maxspeed × Length Max flow Core (ml) (cm) (cm) 1000 RPM (cm) (ml/min) Coreof 3200 5.5 6.6 40.5 76.2 667 Core of 1600 5.5 6.6 40.5 38.1 333 Core of800 5.5 6.6 40.5 38.1 333 Core of 400 5.5 6.6 40.5 38.1 333

Example 9 Calculations and Results

Run parameter calculations were made starting with calculation of therelative centrifuge of force (g): $\begin{matrix}{{{RCF}(g)} = {( {1.421 \times 10^{- 5}} )\quad({RPM})^{2}d}} \\{\quad{{= {2.3307953 \times 10^{- 4}\quad d}},}} \\{d\quad\quad\text{-}{rotor}\quad{diameter}\quad{in}\quad{{inches}.}} \\{{RPM}\quad\text{-}\quad{speed}\quad{in}\quad{revs}\quad{per}\quad{minute}}\end{matrix}$minute

Core of FIG. 6:

-   -   Core (4.289 diameter)=g=99,967.81    -   Rotor assembly (5.201 diameter)=g=121,224.66.

The K factors, runtimes and flow rates were determined as follows:

Determination of K Factor:${K\quad{Factor}} = \frac{( {2.53 \times 10^{5}} )\quad L_{N}\quad( {R_{MAX}/R_{MIN}} )}{( {{RPM}\text{/}1000} )^{2}}$

For example, the K Factor the core of FIG. 6 running at 40.5 k RPM iscalculated as: $\begin{matrix}{K = \frac{( {2.53 \times 10^{5}} )\quad L_{N}\quad( {2.60/2.14} )}{1.6402 \times 10^{3}}} \\{K = \frac{4.92605 \times 10^{4}}{1.6402 \times 10^{3}}} \\{K = 29.74}\end{matrix}$

Determination of Run Time

FOR a 700S particle in the core depicted in FIG. 6:

-   -   K=30    -   T=K/S (Time required to pellet the virus)    -   T−30/700=0.043 HRS=2.58 MINS

It is understood that 700 is the approximate sedimentation coefficientof the product.

The assembly within which the core of FIG. 6 is housed is 3.2 litersminus the amount of gradient.

Determination of Flow Rates

The flow rates for each separation were calculated for the followingcores:

Typical separation flow rates. Typical separation flow rates. Flow Timeto Through Core Sediment Residence Time Volume Flow Rate 2.55 min 3.4min 1600 ml  28 L/h FIG. 17 at 2.55 min 3.4 min  800 ml  14 L/h 1600 mlFIG. 18 at 800 ml 2.55 min 3.4 min  400 ml   7 L/h FIG. 19 at 2.55 min3.4 min  200 ml 3.5 L/h 400 ml

The flow rate for sedimentation was determined with gradient at 500ml/min (30 L/hr). The flow transient time was 2.4 min. At 400 ml/min (24L/hr), the transient time was 3 minutes (sufficient time to pellet theproduct).

In all runs involving the large-scale and pilot-scale separations, thefollowing parameters were chosen: 60% Sucrose w/w filled to half therotor volume, run speed 40,500 rpm, flow volume bands for, at a minimum,30 minutes, typically 45 to 60 minutes, collection and sucrose loadingat 25% of product loading flow rate, fractionation into 30 aliquots.

The flow rate for loading and the product collection was determined fromthe run speed and the product, a dilution of the beads in water (to<0.04 OD A₂₆₅) was made and this volume loaded at maximum speed of therotor assembly. Post banding, the rotor was run to rest, fractionscollected and subsequent analysis of the fractions were plotted asrepresented in FIG. 26.

FIG. 26 shows that the banding time was equivalent per run of each ofthe large-scale and pilot scale centrifuges (45 to 60 min). The durationof the run was approximately 30 mins for the flow through, as the volumeof product was approximately 3× the rotor volume. As the data in FIG. 26indicates, the same separation was obtained for all volume formats forboth large-scale and pilot scale systems. Further, a narrow product bandat a similar place in the gradient was observed. The narrow peak was afunction of the efficiency of separation and the bead size distribution,which is possibly smaller than for a viral particle having degradationproducts.

In terms of the gradient formed, half the rotor was loaded as densitymaterial and the recovery shows half the volume contained gradient. Thesucrose loaded as a step has formed a linear format across the rotor. Atthe maximum density, a sharp cut off was seen. A drop in density wasalso observed where back mixing occurred due to residual amounts ofbuffer introduced to the tubing during the continuous flow portion ofthe run.

Theoretical sedimentation, which was achieved in all cases during thepredicted time, was seen to be marginally incomplete as a tail wasobserved on each product peak.

Analysis of product peaks for each run indicates similar peak height andwidth in both the large-scale and pilot scale centrifuge systems. Thepeak density was similar in all centrifuges and any variation was afunction of the fractionation pattern by 1 or 2 fractions as seen in thetable below. Peak analysis for each separation Peak Peak DensityRecovery Recovery Range @ @ 25% @ 25% Peak 25% threshold threshold PeakFraction Density threshold Density Range Core A₂₈₀ A₂₆₅ (sucrose %)(g/cm³) (sucrose %) (g/cm³) Core of  83% 82 41 1.1816 38-411.1663-1.1816 Core of 79 86 43 1.1920 39-43 1.1713-1.1868 with 1600 mlavailable Core of 70 70 42 1.1868 38-42 1.1663-1.1868 with 800 mlavailable. Core of 85 94 42 1.1868 33-46 1.1415-1.2079 with 400 mlavailable.

Analysis of the gradient slope by both polynomial analysis and linearregression analysis, as identified below, indicates that there is asubstantially identical fit (R2 value). Further, each gradient formed tothe same shape, as indicated by the polynomial fit curve. Further, thesecharts also show that the product separating section of the gradient wasequivalent by the linear application of regression equation (over 25 to50% w/w sucrose) at that point. All of the preceding confirms, in otherwords, that linearity and scalability are achieved. Slope of gradients.Polynomial Analysis Core Equation R2 y = −0.1636x² + 9.8708x − 86.211 R2= 0.9975 FIG. 17 at 1600 ml y = −0.245x² + 12.342x − 97.675 R2 = 0.9952FIG. 18 at 800 ml y = −0.2059x² + 9.5983x − 53.195 R2 = 0.9292 FIG. 19at 400 ml y = −0.2675x² + 15.573 − 177.22 R2 = 0.9346

Slope of gradients. Linear Regression Analysis Core Equation R2 y =3.4405x + 21.393 R2 = 0.9926 FIG. 17 at 1600 ml y = 3.25x + 22.545 R2 =0.9929 FIG. 18 at 800 ml y = 4.1845x + 21.982 R2 = 0.9979 FIG. 19 at 400ml y = 3.65x + 22.861 R2 = 0.9981

FIG. 26 shows that a similar gradient shape is achievable with theembodiments of the present invention. Further, and as indicated in thetables above, the slope of the gradients formed, determined by bothpolynomial analysis and linear regression, have near-identical R2values. In other words, from FIG. 26 and the analyses of the gradientslope, the present invention achieved both scalability and linearity ofthe particle separations by, for example, altering the fin dimensionsand, thereby, altering the volume of the core. This indicates that thegradient remains identical despite the volumetric difference betweeneach separation. These examples demonstrate, inter alia, that acentrifuge apparatus and process in which the volume of the productsample centrifuged can be scaled up or down while maintainingsubstantially the same selected separation parameters of the process;that a centrifuge apparatus and process in which the volumetric capacityof the rotor assembly of the centrifuge can be varied or changed toaccommodate different volumes of product sample to be centrifuged; andthat replaceable cores of different sizes can be utilized in the samecentrifuge apparatus to change the volumetric capacity of the rotorassembly to allow scale ups or scale downs of product sample to becentrifuged without substantially altering selected separationparameters such as sedimentation path, residence path and flow dynamics.

Thus, these examples demonstrate that both scalability and linearity areobtainable. Scalability was demonstrated because the run parametersremained substantially the same, even though rotor assembly volume wasvaried by varying the dimensions of the fins 13. Further, and as shownin FIG. 26 and the tables above wherein substantially equivalent R2values were observed by both polynomial analysis and linear regressionanalysis, these examples demonstrate that linearity is obtainablebecause equivalent gradient formation among the large-scale and pilotscale rotor assemblies was achieved; equivalent product separation atthe iso-dense layer in each scale of rotor assembly was achieved; andequivalent product peak shape in the gradient for each scale rotorassembly was achieved.

Although preferred embodiments of the present invention andmodifications thereof have been described in detail herein, it is to beunderstood that this invention is not limited to those preciseembodiments and modifications, and that other modifications andvariations may be affected by one skilled in the art without departingfrom the spirit and scope of the invention as defined by the appendedclaims.

1. A centrifuge apparatus operable at certain predetermined parametersdepending upon a product to be separated and is useable with a pluralityof rotor assemblies wherein a first rotor assembly of said plurality ofrotor assemblies includes a first core having a first core configurationwhich is contained within a rotor housing of the first rotor assembly todefine a first volume capacity such that the product passing through thefirst rotor assembly having the first volume capacity during rotation ofthe first rotor assembly in the centrifuge apparatus achieves a firstparticle separation of the product, and a second rotor assembly of saidplurality of rotor assemblies includes a second core having a secondcore configuration which is contained with a rotor housing of the secondrotor assembly to define a second volume capacity such that productpassing through the second rotor assembly having the second volumecapacity during rotation of the second rotor assembly in the centrifugeapparatus achieves a second particle separation of the product which isa linear change with respect to the first particle separation.
 2. Thecentrifuge apparatus of claim 1, wherein the rotor housing of the firstand the second rotor assemblies is the same rotor housing.
 3. Thecentrifuge apparatus of claim 1, wherein the rotor housings of the firstand second rotor assemblies have the same residence length.
 4. Acentrifuge apparatus operable at certain predetermined parametersdepending upon a product to be separated and is usable with a pluralityof rotor assemblies wherein a first rotor assembly of said plurality ofrotor assemblies has a first residence length such that the productpassing through the first rotor assembly during rotation thereof in thecentrifuge apparatus achieves a first particle separation of the productand a second rotor assembly of said plurality of rotor assemblies has asecond residence length such that the product passing through the secondrotor assembly during rotation thereof in the centrifuge apparatusachieves a second particle separation of the product which is a linearchange with respect to the first particle separation.
 5. A method forachieving linear scale separation of particles of a product duringcentrifugation comprising the steps of: operating a centrifuge apparatusat certain predetermined parameters depending upon a product to beseparated; placing a first core having a first core configuration in arotor housing to define a first rotor assembly having a first volumecapacity; rotating the first rotor assembly having the first volumeassembly having the first volume capacity in the centrifuge apparatusand passing the product through the first rotor assembly during rotationthereof so as to achieve a first particle separation of the product;substituting a second core having a second core configuration within therotor housing to define a second rotor assembly having a second volumecapacity; and rotating the second rotor assembly having the secondvolume capacity in the centrifuge apparatus and passing the productthrough the second rotor assembly during rotation thereof so as toachieve a second particle separation of the product which is a linearchange with respect to the first particle separation.
 6. A method forachieving linear scale separation of particles of a product duringcentrifugation comprising the steps of: operating a centrifuge apparatusat certain predetermined parameters depending upon a product to beseparated; rotating a first rotor assembly having a first residencelength in the centrifuge apparatus; passing the product through thefirst rotor assembly during rotation thereof to achieve a first particleseparation of the product; substituting the first rotor assembly in thecentrifuge apparatus with a second rotor assembly having a secondresidence length and rotating the second rotor assembly within thecentrifuge apparatus; and passing the product through the second rotorassembly during rotation thereof to achieve a second particle separationof the product which is a linear change with respect to the firstparticle separation.
 7. A centrifuge apparatus for separating particlesof a product, said apparatus comprising means for setting a number ofparameters and adjustment means operable at the set parameters andhaving one of a rotor assembly selected from among a plurality of rotorassemblies so as to enable volume capacity to be adjusted.
 8. Thecentrifuge apparatus for separating particles of a product of claim 7,wherein said adjustment means enables substitution of a rotor core ofvarying configurations within each of said plurality of rotorassemblies.
 9. The centrifuge apparatus for separating particles ofproduct of claim 7, wherein each respective rotor core of the pluralityof rotor assemblies includes a plurality of fins arranged in apredetermined manner.
 10. The centrifuge apparatus for separatingparticles of a product of claim 7, wherein the plurality of fins of eachrespective rotor core are equidistantly spaced apart form each other.11. The centrifuge apparatus for separating particles of a product ofclaim 7, wherein between 0 to 36 fins extend radially outwardly from therotor core.
 12. The centrifuge apparatus for separating particles ofclaim 11, wherein between 0 to 6 fins extend radially outwardly from therotor core.
 13. A rotor assembly rotatable in a centrifuge assembly forseparating particles of a product passing therethrough, said rotorassembly comprising: a rotor housing of a defined volume; and a rotorcore freely rotatable within the rotor housing, said rotor coreincluding a plurality of product flow distribution channels and aplurality of fins extending radially therefrom of a predeterminedconfiguration to define a volume of the predetermined rotor core.
 14. Arotor core for a rotor assembly rotatable in a centrifuge assembly forseparating particles of a product passing through the rotor assembly,said rotor core including a plurality of product flow distributionchannels and a plurality of fins extending radially therefrom of apredetermined configuration to define a predetermined volume of therotor core.
 15. The rotor core of claim 14, wherein the fins of saidplurality of fins are equidistantly spaced apart from each other. 16.The rotor core of claim 14, wherein said plurality of fins are between 0to 36 in number.
 17. The rotor core of claim 16, wherein said pluralityof fins are between 0 to 6 in number.